Biorefinery system, methods and compositions thereof

Abstract

The present disclosure relates to bioengineering approaches for producing biofuel and, in particular, to the use of a C 1 metabolizing microorganism reactor system for converting C 1 substrates, such as methane or methanol, into biomass and subsequently into biofuels, bioplastics, or the like.

What is claimed is: 
     
         1 . A biomass oil composition comprising whole and/or lysed cells of methanotroph bacteria, wherein said methanotroph bacteria comprise a heterologous polynucleotide and wherein said methanotroph bacteria comprising said heterologous polynucleotide produce a biomass oil composition comprised of more than about 50% w/w terpenoid or isoprenoid compounds. 
     
     
         2 . The biomass oil composition of  claim 1 , wherein said methanotroph bacteria is selected from the group consisting of  Methylomonas  sp. 16a (ATCC PTA 2402),  Methylosinus trichosporium  OB3b (NRRL B-11,196),  Methylosinus sporium  (NRRL B-11,197),  Methylocystis parvus  (NRRL B-11,198),  Methylomonas methanica  (NRRL B-11,199),  Methylomonas albus  (NRRL B-11,200),  Methylobacter capsulatus  Y (NRRL B-11,201),  Methylococcus capsulatus  Bath (NCIMB 11132),  Methylobacterium organophilum  (ATCC 27,886),  Methylomonas  sp. AJ-3670 (FERM P-2400),  Methylomicrobium alcaliphilum, Methylocella silvestris, Methylacidiphilum infernorum, Methylibium petroleiphilum, Methylobacterium populi , and any combination thereof. 
     
     
         3 . The biomass oil composition of  claim 1 , wherein said methanotroph bacteria comprises a heterologous polynucleotide encoding a farnesene synthetic enzyme, and said methanotroph bacteria accumulates an increased level of farnesene as compared to a wild-type methanotroph bacteria without said heterologous polynucleotide and grown under same conditions. 
     
     
         4 . The biomass oil composition of  claim 1 , wherein said heterologous polynucleotide encoding said farnesene synthetic enzyme is codon optimized for expression in said methanotroph bacteria. 
     
     
         5 . The biomass oil composition of  claim 1 , wherein said biomass oil composition comprises more than about 50% w/w isoprenoid compounds. 
     
     
         6 . The biomass oil composition of  claim 1 , wherein said biomass oil composition comprises more than about 50% w/w terpenoid compounds. 
     
     
         7 . The biomass oil composition of  claim 6 , wherein said terpenoid compound is farnesene, limonene or both. 
     
     
         8 . The biomass oil composition of  claim 6 , wherein said terpenoid compound is farnesene. 
     
     
         9 . The biomass oil composition of  claim 6 , wherein said terpenoid compound is limonene. 
     
     
         10 . A biomass oil composition comprising (a) a culture of the methanotroph bacteria of  claim 1  together with a culture media in which said methanotroph bacteria were grown; (b) the methanotroph bacteria of  claim 1  grown and recovered from said culture media; or (c) a spent media composition recovered from said culture of said methanotroph bacteria of (a). 
     
     
         11 . The biomass of  claim 10 , wherein said biomass oil composition is said spent media composition recovered from said culture of said methanotroph bacteria of (a), and further comprising an oil composition that is extracted or concentrated from said spent media composition. 
     
     
         12 . A method of producing a biomass oil composition, comprising culturing methanotroph bacteria in a controlled culturing unit in the presence of a feedstock comprising a C 1  substrate under conditions and for a time sufficient to produce a biomass oil composition, wherein said methanotroph bacteria comprise a heterologous polynucleotide and wherein said biomass oil composition comprises whole and/or lysed cells of methanotroph bacteria and more than about 50% w/w terpenoid or isoprenoid compounds. 
     
     
         13 . The method of  claim 12 , wherein said methanotroph bacteria are cultured in a controlled culture unit selected from the group consisting of a fermentor, a bioreactor, a hollow fiber cell, a packed bed bioreactor, and combinations thereof 
     
     
         14 . The method of  claim 12 , wherein said feedstock comprising a C 1  substrate is natural gas or methane. 
     
     
         15 . The method of  claim 12 , wherein said biomass comprises (a) a culture of said methanotroph bacteria together with a culture media in which said methanotroph bacteria were grown; (b) said methanotroph bacteria recovered from said culture media; or (c) a spent media composition recovered from said culture media comprising said methanotroph bacteria. 
     
     
         16 . The method of  claim 12 , wherein said methanotroph bacteria is selected from the group consisting of  Methylomonas  sp. 16a (ATCC PTA 2402),  Methylosinus trichosporium  OB3b (NRRL B-11,196),  Methylosinus sporium  (NRRL B-11,197),  Methylocystis parvus  (NRRL B-11,198),  Methylomonas methanica  (NRRL B-11,199),  Methylomonas albus  (NRRL B-11,200),  Methylobacter capsulatus  Y (NRRL B-11,201),  Methylococcus capsulatus  Bath (NCIMB 11132),  Methylobacterium organophilum  (ATCC 27,886),  Methylomonas  sp. AJ-3670 (FERM P-2400),  Methylomicrobium alcaliphilum, Methylocella silvestris, Methylacidiphilum infernorum, Methylibium petroleiphilum, Methylobacterium populi , and any combination thereof. 
     
     
         17 . The method of  claim 12 , wherein said methanotroph bacteria are cultured in a bioreactor comprising balanced media. 
     
     
         18 . The method of  claim 12 , wherein said methanotroph bacteria are cultured in a bioreactor comprising unbalanced media having limiting quantities of phosphorus, nitrogen, trace elements, oxygen relative to balanced media, or any combination thereof. 
     
     
         19 . The method of  claim 12 , wherein said biomass oil composition comprises more than about 50% w/w isoprenoid compounds or comprises more than about 50% w/w terpenoid compounds. 
     
     
         20 . The method of  claim 19 , wherein said terpenoid compound is farnesene, limonene or both. 
     
     
         21 . A methanotroph bacteria, wherein said methanotroph bacteria comprises a heterologous polynucleotide encoding an enzyme capable of promoting the production of terpenoid or isoprenoid compounds, and wherein said methanotroph bacteria accumulates an increased level of terpenoid or isoprenoid compounds when grown on a C 1  substrate as a carbon source when compared to a wild-type methanotroph bacteria without said heterologous polynucleotide and grown under the same conditions. 
     
     
         22 . The methanotroph bacteria of  claim 21 , wherein said methanotroph bacteria comprise more than about 50% w/w terpenoid compounds. 
     
     
         23 . The methanotroph bacteria of  claim 21 , wherein said terpenoid compound is farnesene, limonene or both. 
     
     
         24 . A methanotroph bacteria, wherein said methanotroph bacteria comprises a heterologous polynucleotide encoding an enzyme capable of promoting the production of isobutanol, and wherein said methanotroph bacteria accumulates an increased level of isobutanol when grown on a C 1  substrate as a carbon source when compared to a wild-type methanotroph bacteria without said heterologous polynucleotide and grown under the same conditions.